Acidic organelles are present in all cells and tissues of mammalian, plant, yeast and fungal cells, except red blood cells. Many bacteria also contain acidic compartments. These acidic organelles are often involved in metabolism and catabolism of foreign molecules that are brought into the cell by endocytosis. They are often the first line of defense against foreign bacterial or viral infection. The acidic pH of endosomes is critical to the process by which lipid-enveloped viruses enter the cytoplasm after their cellular uptake by receptor-mediated endocytosis. Phagocytosis is the process where extra cellular particles such as bacteria, are engulfed in the cell and then fused to lysosomes for digestion. Acidic organelles have also been shown to be responsible for digestion of high molecular weight proteins, oligosaccharides, glycolipids or peptides by the cell. In addition, they are often involved in therapeutic drug metabolism. Among the cellular organelles that have been found to mediate their enzyme activities by acidification are lysosomes, acidic endosomes, phagosomes, clathrin-coated vesicles and Golgi vescicles.
Lysosomes are an example of an acidic cytoplasmic organelle. Lysosomes have been found to be involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. To date, more than 50 acidic hydrolytic enzymes have been identified that are involved in ordered lysosomal degradation of proteins, lipids, carbohydrates and nucleic acids. Functional deficiencies in these lysosomal enzymes, however, are indicative of a number of disease states.
Many inherited carbohydrate metabolic diseases, especially lysosomal storage diseases, have been identified to date. These diseases include Hurler disease (MPS IH, i.e., mucopolysaccharidosis type IH), Scheie disease (NIPS IS), Hurler-Scheie disease (MPS I H/S), Hunter disease (MPS II), Sanfilippo disease (MPS III), Morquio disease (MPS IV), Maroteaux-Lamy disease (MPS VI), Sly disease (MPS VIII), mannosidosis, fucosidosis, sialidosis, asparylglycosaminuria, Gaucher disease (glucosylceramide lipidosis), Krabbe disease (galactoceramide-lipidosis), Fabry disease, Schindler disease, GM1 gangliosidoses, GM2 gangliosidoses, Tay-Sachs disease, Sandhoff disease, and mucolipidoses. As a group, these diseases are the most prevalent genetic abnormalities of humans. Gaucher disease, Sandhoff disease, Krabbé disease, and Tay-Sachs syndrome comprise the majority of patients in this category and are categorized as sphingolipidoses in which excessive quantities of undegraded fatty components of cell membranes accumulate because of inherited deficiencies of specific catabolic enzymes within lysosomes.
The therapeutic options for treating these diseases are relatively limited; in fact, there are currently no available therapies for many of these disorders. To date, therapeutic efforts have mainly focused on strategies for augmenting enzyme concentrations to compensate for the underlying defect. For this reason, new, sensitive and specific assays for monitoring lysosomal enzyme activities in living cells that will be of significant value in monitoring the success of current therapies and for discovery of new therapeutic strategies for diseases of lysosomal origin are needed.
Traditional lysosomal stains include the non-specific phenazine and acridine derivatives, neutral red and acridine orange, that are accumulated in the acidic vesicles upon being protonated. Fluorescently labeled latex beads and macromolecules, such as dextran, can also be accumulated in lysosomes by endocytosis in a variety of experiments.
Prior stains, methods and assays for visualizing acidic organelles such as lysosomes are not useful for monitoring lysosomal enzyme activities in living cells. For example, weakly basic amines have been shown to selectively accumulate in cellular compartments with low internal pH. When further linked to chromogenic or fluorogenic probes, they can be used to label these compartments. Among these is the frequently used acidotropic probe, N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride (hereafter referred to as DAMP). DAMP is not itself fluorescent and fixation and permeabilization of the cell, followed by the use of anti-DNP antibodies conjugated to a fluorophore, an enzyme or ferritin are required in order to visualize the staining pattern. The fluorescent dyes neutral red and acridine orange are also commonly used for staining acidic organelles, but they lack specificity and are not well retained in the organelles, particularly after fixing and permeabilization.
The compounds dansyl cadaverine and monodansyl cadaverine, which contain an aliphatic amino groups for targeting to the lysosome have been described as a lysosomotropic reagents. However, dansyl cadaverine is only described as having an effect on the function of human natural killer cells and human polymorphonuclear leucocytes. More recent research describes monodansyl cadaverine as a fluorescent label, however it is described as useful as a label for autophagic vacuoles, as it fails to label either endosomal compartments or lysosomes. In addition, the dansyl fluorophore is excited in the ultraviolet region (<350 nm), which is generally incompatible with living systems, has a low quantum yield and has a low extinction coefficient (less than 5,000) requiring high concentrations of dye when staining cells.
In addition, certain dipyrrometheneboron difluoride fluorophores linked to a weak base that is only partially protonated at neutral pH as described in U.S. Pat. No. 5,869,689, have been used for general labeling of lysosomes. But none of these probes are useful for monitoring specific enzyme activities in lysosomes or for metabolic analyses or analysis of enzyme activity defects.
Of the numerous lysosomal storage assay systems that have been reported, the majority utilize either fluorescent (4-methylumbelliferyl) substrates, chromogenic (nitrophenolic glycosides), glycolipids labeled with fluorescent dyes or radioactive substrates for detection of lysosomal glycosidase activities. These methods, however, utilize either cell lysate from cells or tissue homogenates, HPLC separation of enzymatic products and UV or fluorescent analysis or other complex analysis techniques. None of these assays, therefore, are well designed for an in vivo, or live-cell high-throughput systems detection, and require either biopsy or extensive cell preparation steps. In addition, the fluorescent dyes used in these assays are not amenable to the low pH environment of the lysosome and therefore do not allow imaging in the lysosome in its native environment. Accordingly, no methods have thus far been described that employ intact lysosomes or a live-cell format to monitor lysosomal enzyme activities.